Several characteristics permit the identification and discrimination of different cells obtained from a variety of sources by cell type for diagnostic and research purposes. Such characteristics include the size of the cells, the forward light scattering properties of the cells, the 90° light scattering of the cells, the number and type of acidophilic or basophilic granules contained in the cells, the amount of RNA contained in the cells, the condensation of the nuclear chromatin of the cells, the cytoplasm to nuclear ratio of the cells, the physical appearance of nucleoli of the cells, and the number of mitochondria contained in the cells, among others.
The classical use of fluorescent dyes to discriminate cells, especially cells found in whole blood, is usually associated with staining the cell's nucleus or DNA. Many cells have the same amount of DNA, and if such cells are stained using a fluorescent dye, the DNA in each cell is similarly stained. Therefore, discriminating between different blood cells, for example, including lymphocytes, monocytes, neutrophils, eosinophils, basophils, and nucleated red blood cells, among others, is difficult, if not impossible.
There are several fluorescent dyes that have been used to analyze cells and include metachromatic dyes. Metachromatic dyes have excitation and emission wavelengths that shift depending on the manner in which the compound or cell that binds the dye. For example, a metachromatic dye can emit at one wavelength when it binds RNA and at another wavelength when it binds DNA. One common metachromatic dye is Acridine Orange dye (AO), which, when bound to double stranded DNA, has an absorption maximum of 502 nm and an emission maximum of 520 to 524 nm. When AO is bound to single stranded DNA or RNA, it has an absorption maximum of 526 to 558 nm and an emission maximum of 630 to 644 nm. AO also can accumulate in the lysosome granules where it has an emission wavelength of about 630 nm. AO is also known to accumulate in the alpha granules of platelets and the granules of mast cells.
The separation and identification of wavelength differences in cellular identification using metachromatic dyes has been observed (Melamed et al. 1972 Amer. J. Clin. Path., 57:95-102). Specifically, in nucleated cells, the ability to resolve metachromatic staining of nucleated cells at the red wavelengths decreased or was lost, i.e., the 520 to 524 nm emission maximum overlapped into the weaker red fluorescence of the RNA or granules. These authors hypothesized that the same was due to the large amount of DNA staining, the intense fluorescence obtained therefrom, and broad emission spectrum obtained. For reticulocytes, weak RNA staining was superimposed on the nonspecific interaction and fluorescence of the dye into cellular material.
The Acridine Orange metachromatic dye was also used to detect reticulocytes using the red fluorescence and forward scatter to discriminate a cell from noise and to distinguish platelets from reticulocytes and red blood cells. See U.S. Pat. No. 4,325,706.
The difficulties in using dyes including AO are known by those of skill in the art. See, “Flow Cytometry and Cell Sorting” Acridine Orange: A Versatile Probe of Nucleic Acids and Other Cell Constituents; 2nd Ed., Wiley-Liss, Inc.: pages 291-314 (1990). Specifically, it is known that metachromatic dyes such as AO, do not follow the simple laws of mass action in their staining. The dye often causes a phase transition (condensation or precipitation) in binding to the target material. Further, such dyes require precise control of dye concentration, free dye concentration, cell concentration, pH, time, and temperature to obtain reproducibility. Precise control is difficult or impossible to obtain in routine applications.
Equilibrium staining methods employing AO have been used for DNA/RNA content measurement, but are noted to be difficult to apply to intact cells and impractical for use with fluorescent antibodies or ligands for demonstration of cell surface structures. Therefore, others have turned to combinations of other dyes, such as Hoechst 33342 and pyronin Y (i.e., a xanthine analog of AO) at a 2:1 to 1:1 ratio, and Hoechst 33342 and AO at a 5:1 ratio, to stain intact cells for DNA/RNA content with a dual source flow cytometer (H. M. Shapiro, 1981 Cytometry, 2(3):143-150). However, unstable results were reportedly obtained with the AO combination. These authors reported an accurate estimate of DNA content and biased estimate of RNA content with the Hoechst 33342/pyronin Y combination.
There remains a need in the art for dyes and dye combinations suitable for differential staining fluorescent dyes and/or combinations thereof for effective cellular identification.